Serotonin receptor‐mediated vasorelaxation occurs primarily through 5‐HT4 activation in bovine lateral saphenous vein

Abstract To better understand mechanisms of serotonin‐ (5‐HT) mediated vasorelaxation, isolated lateral saphenous veins from cattle were assessed for vasoactivity using myography in response to increasing concentrations of 5‐HT or selective 5‐HT receptor agonists. Vessels were pre‐contracted with 1 × 10−4 M phenylephrine and exposed to increasing concentrations of 5‐HT or 5‐HT receptor agonists that were selective for 5‐HT1B, 5‐HT2B, 5‐HT4, and 5‐HT7. Vasoactive response data were normalized as a percentage of the maximum contractile response induced by the phenylephrine pre‐contraction. At 1 × 10−7 M 5‐HT, a relaxation was observed with an 88.7% decrease (p < 0.01) from the phenylephrine maximum. At 1 × 10−4 M 5‐HT, a contraction was observed with a 165% increase (p < 0.01) from the phenylephrine maximum. Increasing concentrations of agonists selective for 5‐HT2B, 5‐HT4, or 5‐HT7 resulted in a 27%, 92%, or 44% (p < 0.01) decrease from the phenylephrine maximum, respectively. Of these 5‐HT receptor agonists, the selective 5‐HT4 receptor agonist resulted in the greatest potency (−log EC50) value (6.30) compared with 5‐HT2B and 5‐HT7 receptor agonists (4.21 and 4.66, respectively). To confirm the involvement of 5‐HT4 in 5‐HT‐mediated vasorelaxation, blood vessels were exposed to either DMSO (solvent control) or a selective 5‐HT4 antagonist (1 × 10−5 M) for 5‐min prior to the phenylephrine pre‐contraction and 5‐HT additions. Antagonism of the 5‐HT4 receptor attenuated the vasorelaxation caused by 5‐HT. Approximately 94% of the vasorelaxation occurring in response to 5‐HT could be accounted for through 5‐HT4, providing strong evidence that 5‐HT‐mediated vasorelaxation occurs through 5‐HT4 activation in bovine peripheral vasculature.


| INTRODUCTION
Consumption of ergot alkaloids found in wildtype endophyte-infected (Epichlöe coenphialum) tall fescue (Lolium arundinaceum) can lead to fescue toxicosis, a form of ergotism in ruminant livestock species (Bush et al., 1982;Lyons et al., 1986;Strickland et al., 2011).Increased core temperature, rectal temperature, and respiration rate, and decreased vascular cross-sectional area, blood flow, and heart rate are common clinical symptoms of increased vasoconstriction in cattle consuming ergot alkaloids from toxic endophyte-infected tall fescue (Aiken et al., 2007(Aiken et al., , 2009;;Al-Haidary et al., 2001;Koontz et al., 2012;Rhodes et al., 1991).Prolonged display of clinical symptoms in response to fescue toxicosis could potentially manifest into impaired cardiovascular function and thermoregulation, peripheral tissue necrosis, and intrauterine growth restriction (Duckett et al., 2014;Strickland et al., 2011).Mechanisms describing how ergot alkaloid ingestion relates to the development of clinical symptoms of fescue toxicosis are not completely understood.Ergot alkaloids are structurally similar to monoamine neurotransmitters such as serotonin (5-hydroxytryptamine; 5-HT), dopamine, epinephrine, and norepinephrine (Berde, 1980), and thus can interact with their respective receptors to cause numerous effects on animal physiology and metabolic function (Klotz, 2015).
It is well-established that vascular contraction and relaxation responses to 5-HT can vary depending on the animal species, type of blood vessel, and presence of 5-HT receptor subtypes (Ni & Watts, 2006).A common finding between studies is that 5-HT can mediate vasorelaxation in isolated veins but not in isolated arteries, unless the arteries are pre-contracted and receptors mediating contractile responses are pharmacologically antagonized (Davis et al., 2012).Vasorelaxation of isolated veins in response to 5-HT has been demonstrated in numerous species and across different tissues (Chand, 1981;Cocks & Arnold, 1992;Gupta, 1992;Ishine et al., 2000;Leung et al., 1996;Seitz et al., 2017;Trevethick et al., 1984;Tsuru et al., 1998;Zhang et al., 1995).Serotoninmediated vasorelaxation of the isolated feline saphenous vein has been previously demonstrated (Feniuk et al., 1983).However, it is currently unknown which 5-HT receptor(s) mediate vasorelaxation in the isolated bovine lateral saphenous vein (Watts, 2016).The objectives of the current study were to determine the relaxant potential of 5-HT and to identify potential 5-HT receptor subtypes mediating vasorelaxation activity in bovine lateral saphenous vein.

| MATERIALS AND METHODS
No live animals were involved in this study, thus approval from the University of Kentucky Animal Care and Use Committee was not required.

| Blood vessel collection
Cranial branches of the lateral saphenous vein were collected (Klotz et al., 2006) from the left and right hind legs of six steers for Experiment 1 (body weight = 507 ± 59.5 kg) and four heifers for Experiment 2 (body weight = 650 ± 52.0 kg) immediately after slaughter at the University of Kentucky Meats Laboratory.Steers used in Experiment 1 were predominantly of Angus breeding (n = 5) and one steer was Holstein.The heifers used in Experiment 2 were predominantly of Angus breeding.Blood vessels were collected over a 22-day period in Experiment 1 and a 16-day period in Experiment 2. Collected vessel samples with surrounding adipose and connective tissues were immersed in oxygenated modified Krebs-Henseleit buffer (95% O 2 /5% CO 2 ; pH = 7.4; 11.1 mM d-glucose; 1.2 mM MgSO 4 ; 1.2 mM KH 2 PO 4 ; 4.7 mM KCl; 118.1 mM NaCl; 3.4 mM CaCl 2 ; 24.9 mM NaHCO 3 ; Sigma-Aldrich, St. Louis, MO, USA), stored on ice, and transported to the laboratory.Blood vessels were dissected, and perivascular adipose and connective tissues were removed.Cleaned vessel segments were sliced into approximately 2-mm cross-sections using an adjustable acrylic tissue matrix (Braintree Scientific, 2006).Vascular dimensions (length, inner diameter, outer diameter) for vein cross-sections were examined using a dissection microscope (Stemi 2000-C, Carl Zeiss Inc., Oberkochen, Germany) at 12.5× magnification and recorded using Axiovision software (version 20, Carl Zeiss Inc.).In addition, vessel cross-sections were inspected under magnification for abnormalities (structural damage incurred during dissection and cleaning and the presence of venous valves and micro-branches) and abnormal sections were discarded and replaced with viable sections.
For Experiment 2, stock solutions of phenylephrine hydrochloride and 5-HT hydrochloride were prepared and diluted as described for Experiment 1.In addition, stock solutions of GR 125487 sulfamate ( 5

| Myography
Three multi-myograph systems (DMT 610M, Danish Myo Technology, Atlanta, GA) containing four individual chambers per myograph were used for each experiment.Prior to calibration, each myograph was turned on to reach the desired temperature setting (37°C).Each chamber of the myograph was calibrated prior to the initiation of the experiment according to the manufacturer's instructions.Blood vessels were mounted onto luminal pins (Wenceslau et al., 2021) and submerged with 5 mL of Krebs-Henseleit buffer and constant gassing (95% O 2 /5% CO 2 ; pH = 7.4; 37°C).The Krebs-Henseleit buffer used in myograph chambers was the same composition of the transport buffer with added 3 × 10 −5 M desipramine (D3900; Sigma-Aldrich) to inactivate neuronal catecholamine reuptake.An equilibration period was conducted under the conditions previously described for 90 min with buffer changes every 15 min to allow blood vessels to reach a resting tension of approximately 1 g.At completion of the 90-min equilibration period, 25 μL of 1 × 10 −4 M phenylephrine was added to each myograph chamber to precontract each vessel segment for 15 min.Following the phenylephrine pre-contraction, 25 μL of 5-HT or selective 5-HT receptor agonists were added to myograph chambers non-cumulatively for 5 min in duplicate for each working concentration.
For Experiment 2, the myography procedure was modified as follows.At completion of the 90-min equilibration period, 25 μL of DMSO or GR 125487 sulfamate (1 × 10 −5 M) was added to myograph chambers for 5-min.Then, 25 μL of 1 × 10 −4 M phenylephrine was added to each myograph chamber to pre-contract each vessel segment for 10 min.Following the phenylephrine pre-contraction, 25 μL of 5-HT was added to myograph chambers noncumulatively for 5 min in duplicate for each working concentration.Standard additions were added to myograph chambers non-cumulatively to avoid potential receptor desensitization (Wenceslau et al., 2021) and to eliminate time as a factor influencing relaxation responses.

| Vasoactive response and potency measurements
Vasoactive responses to treatments were measured for each animal in duplicate and averaged.Isometric changes in tension (measured in millinewtons) of lateral saphenous vein segments in response to phenylephrine, 5-HT, or 5-HT receptor agonists were digitized and converted to mass units (in grams) using PowerLab16/35 and Chart software (version 8.1, ADInstruments, Colorado Springs, CO).A representative trace from Experiment 2 is presented (Figure 1).Baseline tension was determined just prior to the addition of 1 × 10 −4 M phenylephrine.The tension (measured in grams) during the phenylephrine pre-contraction period was determined after plateau (15 min for Experiment 1 and 10 min for Experiment 2) and corrected for baseline tension (Wenceslau et al., 2021).If the greatest change in tension during the 5-min treatment incubation period was increased compared to the pre-contraction maximum, the maximum grams of tension was measured.If the greatest change in tension during the 5-min treatment incubation period was decreased compared to the pre-contraction maximum, the minimum grams of tension was measured.Recorded grams of tension during the 5-min treatment incubation period was normalized as a percentage of the maximum grams of tension induced by the phenylephrine pre-contraction at plateau to compensate for differences in vessel responsiveness.Vessel vasoactive response data were reported as the percent mean vasoactive response (±standard error of the mean) of the maximum contractile response produced by the phenylephrine pre-contraction.A vasoactive response greater than 100% was considered a contraction and a vasoactive response less than 100% was considered a relaxation.Vasoactive response data were plotted using nonlinear regression with fixed slope (GraphPad Prism 9.5; Dotmatics, Boston, MA) and calculated using a log(agonist) vs. response three parameter equation (Wenceslau et al., 2021): where y represents vasoactive response, x represents 5-HT or 5-HT receptor agonist concentration, top and bottom are plateaus in units of the y-axis (percentage of phenylephrine pre-contraction), and 50% effective concentration (EC 50 ) is the molar concentration of 5-HT or 5-HT receptor agonist producing 50% of the response between plateaus.

| Statistical analysis
For Experiment 1, all vasoactive response data were analyzed separately for each compound evaluated using SAS (version 9.4; SAS Institute Inc., Cary, NC).Vasoactive response data were analyzed as a completely randomized design using the GLM procedure for the fixed effect of agonist concentration.For Experiment 2, vasoactive response data were analyzed as a completely randomized design using the GLM procedure for fixed effects of 5-HT 4 receptor antagonist concentration, 5-HT concentration, and the 5-HT 4 receptor antagonist × 5-HT concentration interaction.Least squares means and their standard errors were computed for each fixed effect included in the models.Because of the variable sample size, standard error of the mean (SEM) was used to illustrate the uncertainty surrounding the means.Pairwise comparisons of least squares means were separated using the Tukey-Kramer adjustment, protected by a significant F-test.Results were considered significant if p ≤ 0.05.

| Experiment 2: 5-HT-mediated vasoactivity in the absence and presence of the 5-HT 4 receptor antagonist
The length, inner diameter, and outer diameter of the lateral saphenous vein cross-sections were 2.61 ± 0.154 mm, 0.733 ± 0.140 mm, and 3.09 ± 0.206 mm, respectively.There was a 5-HT 4 receptor antagonist × 5-HT concentration interaction (p < 0.001) for the vasoactive response of the lateral saphenous vein (Figure 7).At 1 × 10 −9 M 5-HT, the vasoactive response did not differ among treatments (with or without the antagonist).
Increasing 5-HT concentrations decreased the vasoactive response below the phenylephrine pre-contraction maximum in the absence of the 5-HT 4 antagonist.In the presence of the 5-HT 4 antagonist, the decrease in the vasoactive response below the phenylephrine precontraction maximum was attenuated and increases in the vasoactive response above the phenylephrine pre-contraction maximum were observed at 1 × 10 −6 M and 1 × 10 −5 M 5-HT.At 1 × 10 −4 M 5-HT, an increase in the vasoactive response above the phenylephrine precontraction maximum was observed in the absence and presence of the 5-HT 4 antagonist and the magnitude of the response did not differ between treatments at this concentration of 5-HT.

| 5-HT stimulates concentration-dependent vasoconstriction and vasorelaxation in the isolated saphenous vein
Early studies identified that 5-HT isolated from porcine and bovine serum functioned as a hypertensive agent and could change vascular tone (Rapport et al., 1948;Watts, 2016;Whitaker-Azmitia, 1999).Several studies have demonstrated that 5-HT causes a concentrationdependent contraction in isolated arteries and veins in multiple species (Page & McCubbin, 1953;Watts et al., 2012).Under most circumstances, 5-HT-induced vasoconstriction is mediated primarily through 5-HT 2A but mediation could also occur through 5-HT 1B , 5-HT 1D , or 5-HT 2B during hypertension (Banes & Watts, 2002;Klotz et al., 2018;Russell et al., 2002).In the current study, increased concentrations of 5-HT induced a contractile response greater than the pre-contracted saphenous vein.This is similar to prior studies in cattle saphenous vein that evaluated 5-HT without a precontraction period (Klotz et al., 2012(Klotz et al., , 2013;;Valente et al., 2021).Notably, the 5-HT concentration curve of the bovine lateral saphenous vein of the current study appears to be similar to the response observed by Watts (Watts, 2016) in the pre-contracted rat tail artery in the presence of 5-HT 2A and 5-HT 1B antagonists.In both studies, 5-HT causes a concentration-dependent relaxation as 5-HT concentrations increased to 1 × 10 −6 M. At concentrations greater than 1 × 10 −6 M, 5-HT caused a vascular contraction.These studies provide greater insight into the dual nature of 5-HT as a vasoconstrictor and vasodilator in isolated blood vessels.
In Experiment 2, increasing 5-HT caused a dosedependent relaxation of the isolated saphenous vein in the absence of the 5-HT 4 receptor antagonist.In the presence of the 5-HT 4 receptor antagonist, nearly all 5-HT-mediated vasorelaxation activity was attenuated.Maximal vasorelaxation was observed at 1 × 10 −7 M 5-HT and 94% of 5-HT-mediated vasorelaxation could be accounted for through 5-HT 4 .Therefore, this experiment confirmed that 5-HT-mediated vasorelaxation occurs primarily through 5-HT 4 activation in the isolated bovine saphenous vein.
Compared with other 5-HT receptor subtypes, there is less information on the role of 5-HT 4 in cardiovascular function.The 5-HT 4 receptor is a seven transmembrane spanning G protein-coupled receptor that exists in two complexes, one coupling G s and one coupling G i proteins (Huang et al., 2022).Several prior investigations into the functional roles of 5-HT 4 described its function in tissues of the central nervous system, gastrointestinal tract, bladder, heart, and adrenal gland (Hegde & Eglen, 1996).Enterokinetic drugs such as prucalopride (Shin et al., 2014;Shokrollahi et al., 2019;Wong et al., 2010), metoclopramide (Bockaert et al., 1990;Elswood et al., 1991;Elz & Keller, 1995), and zacopride (Bhandari & Andrews, 1991;Gullikson et al., 1992) can function as full or partial 5-HT 4 receptor agonists, altering gastrointestinal motility to alleviate chronic constipation and prevent nausea.Notably, Cocks and Arnold (Cocks & Arnold, 1992) were the first to demonstrate vasorelaxation via 5-HT 4 receptor activation in the isolated ovine pulmonary vein.In their study, 5-HT 4 -mediated vasorelaxation appeared to be specific to ruminants because there was no evidence for 5-HT 4 -mediated vasorelaxations of the dog, pig, or human pulmonary vein (Cocks & Arnold, 1992).Other studies with isolated ovine or caprine pulmonary vein have demonstrated 5-HT-induced vasorelaxation but, the involvement of 5-HT 4 was not evaluated (Chand, 1981;Eyre, 1975;Zhang et al., 1995).The results of the current study support the concept that 5-HT 4 -mediated vasorelaxation may be ruminant-specific; however, it remains unknown if the isolated saphenous vein from nonruminant species would relax to 5-HT via 5-HT 4 receptor activation.
There is a lack of clear evidence describing the mechanism by which 5-HT 4 receptor activation leads to relaxation of vascular tissue.Cocks and Arnold (1992) demonstrated that 5-HT 4 -mediated vasorelaxation was endothelium-independent in the isolated ovine pulmonary vein.A later study demonstrated that 5-HT-induced vasorelaxation of the isolated ovine pulmonary vein increased tissue cyclic adenosine monophosphate (cAMP) concentrations and suggested that cAMP was involved in secondary messenger signaling (Zhang et al., 1995).This finding is supported by positive coupling of the 5-HT 4 receptor to adenylate cyclase in other tissues (Bockaert et al., 1990;Dumuis et al., 1988).Further research is necessary to describe the signaling mechanism associated with 5-HT 4 -mediated vasorelaxation of vascular tissue in ruminants.
In conclusion, 5-HT causes both concentrationdependent vasoconstriction and vasorelaxation in the isolated bovine lateral saphenous vein.Vasorelaxation of the bovine saphenous vein can be partially mediated through 5-HT 2B , 5-HT 4 , and 5-HT 7 receptors.Approximately 94% of the maximal vasorelaxation activity occurring in response to 5-HT could be accounted for through 5-HT 4 , providing strong evidence that 5-HT-mediated vasorelaxation occurs primarily through 5-HT 4 receptor activation in bovine lateral saphenous vein.The isolated bovine lateral saphenous vein is an excellent model to further study the functional role of the 5-HT 4 receptor in vascular tissue and signaling mechanisms associated with 5-HT 4 receptor activation.A greater understanding of receptor-mediated vasorelaxation mechanisms could potentially lead to the development of strategies to mitigate sustained ergot alkaloid induced vasoconstriction that occurs during fescue toxicosis in cattle.
AUTHOR CONTRIBUTIONS R.J.T. and J.L.K. conceived and designed the research; R.J.T. and J.L.K. performed the experiments; R.J.T. analyzed the data; R.J.T., D.L.H., and J.L.K. interpreted results of the experiments; R.J.T. prepared the figures; R.J.T.

F
Representative trace of tension in grams from Experiment 2. Downward black arrows indicate the 25 μL addition of (a) dimethyl sulfoxide (DMSO) or (b) selective serotonin receptor 5-HT 4 antagonist [5-HT 4 (-); GR 125487 sulfamate].Downward magenta arrows indicate when baseline tension was recorded and the 25 μL addition of 1 × 10 −4 M phenylephrine to pre-contract the lateral saphenous vein.Downward teal arrows indicate when the maximum grams of tension in response to 1 × 10 −4 M phenylephrine pre-contraction was measured and when the 25 μL addition of 5-HT occurred.Downward purple arrows indicate the end of the 5-min agonist exposure period and when the buffer was evacuated from myograph chambers.The area in between the downward teal and purple arrows is indicated by a black capped line and is where the vasoactive response was measured.When the vasoactive response was lesser than the phenylephrine pre-contraction maximum tension (a), the minimum grams of tension was measured.When the vasoactive response was greater than the phenylephrine pre-contraction maximum tension (b), the maximum grams of tension was measured.
Least squares mean vasoactive response ± SEM (n = 6) of bovine lateral saphenous vein to non-cumulative increasing concentrations of serotonin (5-HT) after pre-contraction with 1 × 10 −4 M phenylephrine.The dotted line at y = 100 represents the normalized vasoactive response to the phenylephrine precontraction.The dashed line at y = 0 represents the normalized vasoactive response at baseline tension.Probability value for 5-HT concentration: p < 0.001.

F
Least squares mean vasoactive response ± SEM (n = 6) of bovine lateral saphenous vein to non-cumulative increasing concentrations of selective 5-HT 1B receptor agonist (CP 93129 dihydrochloride) after pre-contraction with 1 × 10 −4 M phenylephrine.The −log EC 50 was not reported due to the lack of effect of CP 932129 concentration on vasoactive response (p > 0.05).The dotted line at y = 100 represents the normalized vasoactive response to the phenylephrine pre-contraction.Y-axis units do not begin at 0. Probability value for agonist concentration: p = 0.69.F I G U R E 4 Least squares mean vasoactive response ± SEM (n = 6) of bovine lateral saphenous vein to non-cumulative increasing concentrations of selective 5-HT 2B receptor agonist (BW 723C86 hydrochloride) after pre-contraction with 1 × 10 −4 M phenylephrine.The dotted line at y = 100 represents the normalized vasoactive response to the phenylephrine pre-contraction.Y-axis units do not begin at 0. Probability value for agonist concentration: p < 0.001.F I G U R E 5 Least squares mean vasoactive response ± SEM (n = 5) of bovine lateral saphenous vein to non-cumulative increasing concentrations of selective 5-HT 4 receptor agonist (BIMU 8) after pre-contraction with 1 × 10 −4 M phenylephrine.The dotted line at y = 100 represents the normalized vasoactive response to the phenylephrine pre-contraction.The dashed line at y = 0 represents the normalized vasoactive response at baseline tension.Probability value for agonist concentration: p < 0.001.F I G U R E 6 Least squares mean vasoactive response ± SEM (n = 5) of bovine lateral saphenous vein to non-cumulative increasing concentrations of selective 5-HT 7 receptor agonist (LP 44) after pre-contraction with 1 × 10 −4 M phenylephrine.The dotted line at y = 100 represents the normalized vasoactive response to the phenylephrine pre-contraction.Y-axis units do not begin at 0. Probability value for agonist concentration: p < 0.001.